目的：探讨miR-496是否通过靶向mTOR影响宫颈癌HeLa细胞的周期、增殖、迁移、侵袭以及裸鼠移植瘤的生长。方法：选取2020年12月至2021年12月期间于河北医科大学第四医院接受手术的74例宫颈癌患者的肿瘤标本和癌旁组织标本，qPCR、WB法和免疫荧光法检测宫颈癌组织中 miR-496 和 mTOR 在 mRNA 和蛋白水平的表达。利用TargetScan数据库预测miR-496的靶基因并用双荧光素酶报告基因实验进行验证。将HeLa细胞按转染物不同分为对照组、miR-496 mimic组和miR[1]496 mimic+pMIR-mTOR组，CCK-8法、流式细胞术和Transwell实验分别检测miR-496和mTOR对HeLa细胞增殖、周期、迁移和侵袭的影响。将对照组、miR-496 mimic组HeLa细胞接种至BALB/c裸鼠皮下构建宫颈癌裸鼠移植瘤模型，21 d后处死小鼠，剥离小鼠肿瘤组织并称取瘤质量，免疫荧光法和WB法检测miR-496过表达对移植瘤组织中mTOR和Ki67表达的影响。结果：在宫颈癌组织中，miR-496呈低表达，而mTOR mRNA和蛋白呈高表达。miR-496能够靶向结合mTOR mRNA的3’-UTR。与对照组和miR-496 mimic+pMIR-mTOR组相比，miR-496 mimic组HeLa细胞的miR-496水平和S期细胞比例均显著升高，而增殖水平、迁移和侵袭细胞数均显著降低（均P<0.01）。成功构建宫颈癌裸鼠移植瘤模型，接种21 d后，miR-496 mimic组移植瘤质量、移植瘤组织中mTOR和Ki67的表达水平均显著低于对照组（均P<0.01）。结论：miR-496在宫颈癌组织中呈低表达，miR-496过表达可通过靶向调控mTOR抑制HeLa细胞的恶性生物学行为和裸鼠移植瘤的生长。

Objective: To explore whether miR-496 affects the cycle, proliferation, migration, and invasion of cervical cancer HeLa cells and the growth of transplanted tumors in nude mice by targeting mTOR. Methods: The tumor specimens and para-cancerous tissue specimens of 74 patients with cervical cancer who underwent surgery at the Fourth Hospital of Hebei Medical University from December 2020 to December 2021 were selected. qPCR, WB, and immunofluorescence methods were used to detect the expression of miR-496 and mTOR at mRNA and protein levels in cervical cancer tissues. The TargetScan database was used to predict the target genes of miR-496, and the Dual-luciferase reporter gene experiment was further adopted to verify the targeting relationship. According to different transfectants, HeLa cells were divided into the control group, miR-496 mimic group, and miR-496 mimic+pMIR-mTOR group. CCK-8 assay, Flow cytometry, and Transwell experiment were used to detect the effects of miR-496 and mTOR on the proliferation, cell cycle, migration, and invasion of HeLa cells. The HeLa cells of the control group and miR-496 mimic group were inoculated subcutaneously into BALB/c nude mice to construct a transplanted tumor model of cervical cancer. After 21 d, the mice were sacrificed, and the tumor tissues of the mice were stripped and the tumor mass was weighed. Immunofluorescence and WB methods were used to detect the effect of miR-496 overexpression on the expression of mTOR and Ki67 in transplanted tumor tissues. Results: In cervical cancer tissues, miR-496 was lowly expressed, while mTOR was highly expressed at both mRNA and protein levels. miR- 496 could bind to the 3'-UTR sequence of mTOR mRNA. Compared with the control group and the miR-496 mimic+pMIR-mTOR group, the miR-496 level and S-phase cell ratio of HeLa cells in the miR-496 mimic group were significantly increased, while the proliferation level, the numbers of migrated cells and invaded cells were significantly reduced (all P<0.01). Nude mice transplanted tumor model of cervical cancer was successfully constructed. After 21 d of inoculation, the mass of transplanted tumors and the expression of mTOR and Ki67 in the transplanted tumor tissues in the miR-496 mimic group were significantly lower than those in the control group (all P<0.01). Conclusion: miR-496 is lowly expressed in cervical cancer tissues. Overexpression of miR-496 can inhibit the malignant biological behaviors of HeLa cells and the growth of transplanted tumors in nude mice through targeted regulation of mTOR.

河北省医学科学研究重点课题资助项目（No. 20210082）